Polynucleotide encapsulation and preservation using self-assembling membranes

ABSTRACT

Polynucleotides such as DNA are stored inside vesicles formed from self-assembling membranes. The vesicles may be protocells, liposomes, micelles, colloidosomes, proteinosomes, or coacervates. The vesicles may include surface functionalization to improve polynucleotide encapsulation and/or to bind polynucleotides having specific sequences. Encapsulation in vesicles provides protection for the polynucleotides. Additional protection is provided by addition of one or more stabilizers. The stabilizer may be nucleic-acid stabilizers that stabilize the polynucleotides or may be a protective structural layer around the vesicles such as a layer of silica. A process for stably storing polynucleotides in vesicles and a process for recovering stored polynucleotides from vesicles are both disclosed. The polynucleotides may be used for storage of digital information.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a division of U.S. patent application Ser. No.16/177,188, filed Oct. 31, 2018, the content of which is expresslyincorporated herein by reference in its entirety.

BACKGROUND

The volume of digital information is increasing at an exponential rate.This vast increase in the amount of digital information may outpace theability of conventional storage technologies. One promising technologyfor storing large amounts of digital information is deoxyribonucleicacid (DNA). DNA is well known as a molecule that can store geneticinformation. However, DNA can also function as a storage medium fordigital information. Multiple different groups have successfullyconverted computer files into a string of nucleotide bases, synthesizedDNA encoding that string, sequenced the synthetic DNA, and thenrecovered the original digital information with 100% accuracy.

As a storage medium, DNA has potential advantages over conventionaloptical and magnetic media in terms of information densities andstability. Storage using DNA can achieve a theoretical data density ofover 200 petabytes (i.e., 200 million gigabytes) per gram which is muchhigher than possible with conventional media. With DNA it is possiblethat all the digital information in the world could be stored in asingle room. DNA can also provide better long-term storage. Magnetic andoptical media can wear out within five to 10 years. However, readableDNA has been recovered from fossils hundreds of thousands of years old.

Longevity of DNA, however, depends on storage conditions. DNA issusceptible to degradation by heat, enzymes, mutagenic chemicals, andionizing radiation. When stored in solution DNA is stable forapproximately 10 years, which is the same timescale as other storagemedia. Storage techniques that provide long-term stability and highdata-density improve the usability of DNA as a storage medium fordigital information.

SUMMARY

This disclosure provides structures and methods for stably storingnucleotides such as, but not limited to, DNA through encapsulation inself-assembling membranes. The polynucleotides to be stored are mixedwith the precursors of a self-assembling vesicle. A vesicle is a closedstructure formed by amphiphilic molecules (i.e., molecules with bothhydrophilic or “water-loving” and lipophilic, “fat-loving” properties)and may contain a solvent and or other molecules inside the closedstructure. When provided with appropriate conditions (e.g., pH, saltconcentrations, etc.) the precursors spontaneously form sphericalvesicles that contain the nucleotides. These spherical vesicles aresmall: typically on the scale of hundreds of nanometers to tens ofmicrometers (1×10⁻⁷ to 1×10⁻⁵ meters). Types of vesicles that can beartificially formed with self-assembling membranes include protocells,liposomes, micelles, colloidosomes, proteinosomes, and coacervates.

Formation of vesicles in a solution that contains polynucleotides canresult in the polynucleotides being pulled into the inside of thevesicles resulting in the concentration of polynucleotides inside thevesicles being much higher than the concentration in the surroundingsolution. The vesicles' pore size, membrane permeability, interiorfunctionalization, and exterior functionalization can concentrate eitherspecific polynucleotides (i.e., preferentially moving polynucleotideshaving a certain nucleic acid sequence) or non-specific polynucleotidesinto the interior of the vesicle. Functionalization refers to theaddition of specific chemical groups to the surface (interior orexterior) of the vesicle membrane.

Vesicles containing the polynucleotides are concentrated and dried. Thisremoves all or substantially all of the solution and moisture. The driedvesicles may take the form of a powder. Thus, the polynucleotides areconcentrated (relative to the concentration in solution) and protectedby encapsulating in a self-assembling membrane. The polynucleotides arefurther protected by use of one or more stabilizers. The stabilizersprotect the polynucleotides from damage and by doing so increase thelength of time the polynucleotides can be stored without significantdegradation.

Many different types of stabilizers may be used with thevesicle-encapsulated polynucleotides. Some stabilizers act on thepolynucleotides and some act on the vesicles. Some stabilizers providechemical stability and others increase structural stability. Multipledifferent types of stabilizers may be used together. Chemicals such assalts, chelators, or sugars can stabilize the polynucleotide moleculesthemselves. These or similar nucleic-acid stabilizers may be addedduring the formation of the vesicles. Protective layers may also be usedto encapsulate the vesicles. The protective layers can encapsulate thevesicles in a protective shell.

One type of suitable protective coating is silica. Silica, also known assilicon dioxide, has the chemical formula SiO₂ and is the majorconstituent of many types of sand and is the primary ingredient inglass. Silica encapsulation may be performed by exposing the vesicles toa tetra alkoxysilane such as tetraethyl orthosilicate (TEOS).Encapsulation in silica can hermetically seal the vesicles under glassthereby further protecting the polynucleotides from chemical attack,high temperatures, and humidity. Storage conditions may be selected tofurther enhance longevity of the polynucleotides. For example, thepolynucleotides in self-assembling vesicles may be stored at cooltemperatures with low humidity such as, for example, 9° C. and 20%relative humidity. When encased in silica and stored under favorableconditions, the polynucleotides may be preserved without significantdegradation for hundreds, thousands, or potentially millions of years.

Following storage, which may be for tens or hundreds of years, anyprotective layer may be removed from the vesicles with an etchingsolution that dissolves the protective layer without harming thepolynucleotides. For silica encapsulating layers, buffered hydrogenfluoride is a suitable etching solution. If dried, the vesicles arerehydrated and the polynucleotides are released from the vesicles bylysing or disrupting the self-assembling membrane. Once returned tosolution, the polynucleotides may then be sequenced or otherwiseprocessed. When the polynucleotides are used for data storage, thesequence of nucleotide bases in the polynucleotides may be decoded toobtain the stored digital information.

These structures and techniques described in this disclosure haveapplications for storing synthetic polynucleotides encoding digitalinformation but are equally suitable for storing naturally occurringpolynucleotides.

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key features oressential features of the claimed subject matter nor is it intended tobe used to limit the scope of the claimed subject matter. The term“techniques,” for instance, may refer to system(s) and/or method(s) aspermitted by the context described above and throughout the document.

BRIEF DESCRIPTION OF THE DRAWINGS

The Detailed Description is set forth with reference to the accompanyingfigures. In the figures, the left-most digit(s) of a reference numberidentifies the figure in which the reference number first appears. Theuse of the same reference numbers in different figures indicates similaror identical items.

FIG. 1 is a diagram illustrating mixing of polynucleotides and vesicleprecursors to form a self-assembling vesicle that contains thepolynucleotides.

FIG. 2 is a diagram showing the self-assembling vesicle of FIG. 1surrounded by an encapsulating layer.

FIG. 3 flow diagram showing an illustrative process for storingpolynucleotides in self-assembling vesicles.

FIG. 4 is a flow diagram showing an illustrative process for retrievingpolynucleotides that are stored in self-assembling vesicles.

FIG. 5 is a fluorescence microscope image of coacervate-DNA conjugates.

FIG. 6 is a scanning electron microscope (SEM) image of coacervatescovered with a thin film of silicon dioxide.

FIG. 7 is an image of an electrophoretic gel showing that encapsulationwith silica protects DNA from degradation by reactive oxygen species(ROS).

DETAILED DESCRIPTION

Polynucleotides such as DNA and ribonucleic acid (RNA), includingpolynucleotides that have unnatural bases, may be used to store digitalinformation by designing a sequence of nucleotide bases that encodes thezeros and ones of the digital information. There are various techniquesand encoding schemes known to those of skill in the art for usingnucleotide bases to represent digital information. See e.g., Grass etal., Robust Chemical Preservation of Digital Information on DNA inSilica with Error-Correcting Codes, 54 Angew. Chem. Int. Ed. 2552 (2015)and Organick et al., Random access in large-scale DNA data storage, 36:3Nat. Biotech. 243 (2018). Advantages of using DNA rather than anotherstorage media for storing digital information include informationdensity and longevity. The DNA storage structure and methods describedin this disclosure can improve both information density and longevityrelative to other techniques for storing DNA. The contents of thedisclosure may be used with any type of polynucleotide such as DNA, RNA,and DNA-RNA hybrids; thus, references to “DNA” are illustrative and notintended to limit the application to only DNA or to only use of naturalnucleotide bases.

There are many types of damage that can occur to polynucleotides such ashydrolytic damage, oxidative damage, radiation damage, and chemicaldamage. Hydrolytic damage is caused by the presence of water includinghumidity in the air and involves deamination or the total removal ofindividual bases. Polynucleotides stored in water may be subject tobreakdown through enzymatic degradation pathways and also bydepurination. Depurination is a chemical reaction of purinedeoxyribonucleosides, deoxyadenosine and deoxyguanosine, andribonucleosides, adenosine or guanosine, in which the β-N-glycosidicbond is hydrolytically cleaved releasing a nucleic base, adenine orguanine, respectively. When depurination occurs with DNA, it leads tothe formation of AP (apurinic/apyrimidinic) sites that are unstable andpromote strand cleavage. Hydrolytic damage is one of the major sourcesof damage to polynucleotides during storage.

Oxidative damage caused by reactive oxygen species (ROS) is anothermajor source of damage for stored polynucleotides. ROS are chemicallyreactive chemical species containing oxygen. Examples include peroxides,superoxide, hydroxyl radical, singlet oxygen, and alpha-oxygen. Exposureto ROS results in oxidation of DNA strands through the deoxyribose sugarand most prominently to bases with unstable bonds, fragmentation, andcross-coupling. Oxidative DNA damage occurs most readily at guanineresidues due to the high oxidation potential of this base relative tocytosine, thymine, and adenine. 8-hydroxy-deoxyguanosine (8-OHdG) is themost common marker for oxidative DNA damage. High temperature increasesthe chances of strand fragmentation as well as complete pyrolysis.

Ultraviolet and other types of radiation such as cosmic radiation (e.g.,proton or neutron damage) can damage polynucleotides throughfree-radical mechanisms such as strand breaks and cross-linking.Fragmentation or strand breaks involve a cut in one or both DNA or RNAstrands. Radiation-induced damage can also result in the production ofpyrimidine dimers, where covalent cross-links occur in cytosine andthymine residues. These premutagenic lesions alter the structure andpossibly the base-pairing. The most common pyrimidine dimers arecyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidonephotoproducts (6-4PP).

Chemical DNA damage may result from exposure to polycyclic aromatichydrocarbons (PAHs). PAHs are potent, ubiquitous atmospheric pollutantscommonly associated with oil, coal, cigarette smoke, and automobileexhaust fumes. A common marker for DNA damage due to PAHs isBenzo(a)pyrene diol epoxide (BPDE). BPDE is found to be very reactiveand known to bind covalently to proteins, lipids, and guanine residuesof DNA to produce BPDE adducts.

Cold storage methods can reduce the damage that polynucleotidesaccumulate while in storage, but the use of refrigerators, freezers, andliquid nitrogen is expensive and inefficient. Room-temperature storagereduces reliance on bulky and expensive equipment to controltemperature. One technique for storing DNA at room temperature is byplacement of liquid samples on filter paper cards (e.g., Whatman® FTA®cards) with chemicals that lyse cells and stabilize nucleic acids.Another room-temperature storage technique referred to as DNAshell® andRNAshell® (Imagene) places purified and desiccated polynucleotides underan inert gas such as nitrogen, helium, or argon inside airtight,stainless steel capsules. Additionally, there is a recently developedtechnique for adsorbing nucleotides to carbon-functionalized magneticnanoparticles (e.g., TurboBeads®) and encapsulating the nanoparticles insilica as described in U.S. Pat. No. 11,059,016 entitled “SilicaEncapsulated DNA on Magnetic Nanoparticles” filed on Jun. 25, 2018.However, all of the room-temperature techniques described above rely ona physical substrate or container that adds substantial volume andreduces the maximum density of polynucleotide storage. Filter paper,stainless steel capsules, nanoparticles, all contribute their own volumethus using space in storage that could otherwise be occupied bypolynucleotides. This cost to storage density is inherent in any storagesystem that uses containers or substrates.

Stably storing polynucleotides reduces the extent and rate to whichthese and other types of damage occur. The term “stably storing” refersto storage conditions that preserve the items stored in an unchanged orsubstantially unchanged condition for a period of time longer than theitem would be unchanged absent the specific storage conditions. In thecontext of polynucleotide storage, “stably storing” may refer to storingpolynucleotide at room temperature and relative humidity ofapproximately 50% for greater than 10 years with less than 1%degradation. “Stably storing” may also refer to a technique or structurefor storage that reduces degradation of the items stored to a rate thatis less than half that of items stored under similar conditions withoutthe use of the specific storage technique or structure.

FIG. 1 illustrates a schematic representation of a self-assemblingvesicle 100 that may contain polynucleotides 102 and other components.The self-assembling vesicle 100 is generally spherical in shape and iscreated from aggregation of precursors 104 into a membrane 106. Thepolynucleotides 102 may be DNA, RNA, single-stranded (ss),double-stranded (ds), DNA-RNA hybrids, and/or include unnatural bases.The length of the polynucleotides 102 may be, but is not limited to,approximately 80-300 base pairs (bp), approximately 100-200 bp,approximately 120-180 bp, or approximately 150 bp. The polynucleotides102 may encode digital information. The self-assembling vesicle 100 maybe a protocell, a liposome, a micelle, a colloidosome, a proteinosome,coacervate, or another type of vesicle with a biological membrane. Theprecursors 104 are capable of self-assembling to form the membrane 106of the self-assembling vesicle 100. The specific type of precursor 104depends on the type of vesicle 100 and may be native biological lipids,modified lipids, polyamines, polycations, synthetic organic polymers,and/or a combination thereof.

Many of the types of vesicles covered within the scope of thisdisclosure may be referred to as “protocells.” Protocells areself-organized, endogenously ordered, spherical collections of lipidsthat include some functions similar to natural biologic cells. Lipidsthat form protocells are amphipathic molecules that have bothhydrophilic and lipophilic properties. Phospholipids are one type ofamphiphilic molecule and are the main components of biologicalmembranes.

Liposomes, a type of protocell, are spherical vessels with an aqueouscore and at least one lipid bilayer. Liposomes are most often composedof phospholipids, especially phosphatidylcholine, but may also includeother lipids, such as egg phosphatidylethanolamine, so long as they arecompatible with lipid bilayer structure. Liposomes may be multilamellarconsisting of several concentric bilayers, separated by aqueouscompartments or unilamellar, consisting of only one phospholipid bilayersurrounding one aqueous compartment.

Micelles are another type of spherical protocell that differs fromliposomes due to the lack of an aqueous core. A micelle is an aggregate(or supramolecular assembly) of surfactant molecules dispersed in aliquid colloid. A typical micelle in aqueous solution spontaneouslyforms an aggregate with the hydrophilic “head” regions in contact withsurrounding solvent, sequestering the hydrophobic single-tail regions inthe micelle center.

A colloidosome is a microcapsule with a shell that consists ofcoagulated or fused colloid particles. To qualify as a colloid, themixture must be one that does not settle or would take a very long timeto settle appreciably. The dispersed-phase particles have a diameterbetween approximately 1-1000 nm. Colloidosomes may be created asPickering emulsions which an emulsion is stabilized by the addition ofsolid particles such as colloidal silica.

Proteinosomes are a type of protein-based spherical capsules that have asemi-permeable, stimulus-responsive, enzymatically active, elasticmembrane consisting of a closely packed monolayer of conjugated,amphiphilic protein-polymer building blocks. Formation is byself-assembly of a supramolecular protein-polymer conjugate. Oneprotein-polymer conjugate that may be used to form protinosomes isβCD-modified bovine serum albumin (BSA) and adamantane-terminatedpoly(N-isopropylamide) (Ad-PNIPAM) in aqueous solution. Another suitableprotein-polymer conjugate is an aqueous solution of cationized bovineserum albumin (BSA-NH₂)-poly(N-isopropylacrylamide) (PNIPAAm)nanoconjugates. This results in a Pickering emulsion where theamphiphilic BSA-PNIPAAm conjugates self-assemble at the interfaceforming emulsion droplets. A Pickering emulsion is an emulsion that isstabilized by solid particles which adsorb onto the interface betweentwo phases. Chemical cross-linking such as with PEGbis(N-succinimidylsuccinate) can provide additional structural stability. Proteinosomescan be dispersed in oil or water, thermally cycled to temperatures of70° C., and partially dried and re-inflated without loss of structuralintegrity.

Coacervates are spherical aggregates of cationic and anionicwater-soluble polymers that interact in water to form liquid,polymer-rich colloidal droplets held together by hydrophobic forces.Coacervate droplets can measure from 1-100 μm across, while theirsoluble precursors are typically on the order of less than 200 nm.

All the types of vesicles described above have the characteristic offorming substantially instantaneously in solution when the appropriateprecursors 104 are mixed together in the desired ratios under theappropriate conditions such as pH and salt concentration. Additionally,when appropriate precursors 104 are mixed with polynucleotides 102,formation of the vesicles selectively encapsulates the polynucleotides102 resulting in a polynucleotide 102 concentration within theself-assembling vesicle 100 that is higher than the concentration of thepolynucleotides 102 in the surrounding solution. Encapsulation ofpolynucleotides 102 may be promoted by forming the self-assemblingvesicles 100 with a polynucleotide-attracting material encased inside.For example, the vesicles with aqueous cores may be formed in thepresence of streptavidin with oil so that the hydrophilic streptavidinpreferentially remains in the aqueous interior. Biotinylatedpolynucleotides 102 may diffuse through the membrane 106 and anchor tothe encapsulated streptavidin.

The polynucleotides 102 and precursors 104 may be mixed manually or byusing any suitable type of automated liquid-handling equipment such asan automatic pipetting robot or microfluidics system using any type ofsuitable technique for moving liquids such as channel-basedmicrofluidics, digital microfluidics, acoustic droplet ejection (ADE),or other techniques. In an implementation, a first fluid channelcontaining the precursors 104 may be mixed with a second fluid channelthat supplies the polynucleotides 102. The polynucleotides 102 may beprovided in an aqueous solution such as an annealing buffer at aconcentration of, for example, around 20, 30, 40, 50, 60, 70, 80, 90, or100 μg/mL. The polynucleotides 102 provided to the second fluid channelmay be the polynucleotides 102 encoding a particular digital file. Thus,dispensing of the polynucleotides 102 through the automatedliquid-handling equipment may be done on a file-by-file basis orotherwise divided according to the information content of thepolynucleotides 102.

The self-assembling vesicle 100 may be modified with internalfunctionalization 108 and/or external functionalization 110 on theinternal surface or the surface of the external self-assembling vesicle100. The internal functionalization 108 may include the addition offunctional groups that attract the polynucleotides 102. For example,internal functionalization 108 may include a direct targeting mechanismsuch as streptavidin attracting biotinylated polynucleotides 102. Thepolynucleotides 102 are readily biotinylated in the course ofoligonucleotide synthesis by the phosphoramidite method using commercialbiotin phosphoramidite. Other high-affinity binding complexes besidesstreptavidin-biotin may also be used.

Electrostatic charge may be used to attract the polynucleotides 102 tothe interior of the self-assembling vesicle 100. The internalfunctionalization 108 may add a polycationic molecule to the interior ofthe membrane 106 that attracts negatively charged polynucleotides 102.The polycationic molecule may be a polycationic linear macromoleculesuch as polyethyleneimine (PEI), poly-L-lysine (PLL),diethylaminoethyl-dextran (DEAE-dextran), or a branched polymer such aspoly(amidoamine) (PAMAM) dendrimers.

PEI or polyaziridine is a polymer with repeating unit composed of theamine group and two-carbon aliphatic CH₂CH₂ spacer. Linearpolyethyleneimines contain all secondary amines. PLL is a specificchiral configuration of the synthetic polymer α-polylysine with l-lysineat lysine's central carbon. DEAE-dextran is a positively charged dextranderivative that binds and interacts with negatively-charged DNAmolecules and via an unknown mechanism. PAMAM is a class of dendrimerthat is made of repetitively branched subunits of amide and aminefunctionality. PAMAMs have a sphere-like shape overall and are typifiedby an internal molecular architecture including tree-like branching,with each outward layer, or generation, containing exponentially morebranching points. Surface amine residues on PAMAM dendrimers bind to thephosphate backbone of DNA through charged interactions.

Additionally, short oligonucleotide fragments (e.g., ssDNA and/or ssRNA)attached to the interior of the self-assembling vesicle 100 may providesequence-specific attraction for particular polynucleotides 102 due tostrand hybridization. The internal functionalization 108 may add anucleic acid sequence to the inside of the membrane 106 that iscomplementary to the nucleic acid sequence at one of the ends of thepolynucleotides 102. For example, if the polynucleotides 102 aredesigned with primer binding sites at one or both ends, then the primersequence may be used for the internal functionalization 108 toselectively attract those polynucleotides 102 having the primer bindingsite. Synthetic polynucleotides 102 such as those used for storingdigital information will have known sequences and may includeintentionally designed to include primer binding sites that are specificto the type of information encoded. Thus, the internal functionalization108 may cause the self-assembling vesicle 100 to selectively uptake onlya selective subset of the polynucleotides 102 available in solution andthis subset may be only those polynucleotides 102 encoding a specifictype of information (e.g., a particular computer file).

External functionalization 110 adds functional groups to the outside ofthe membrane 106. The external functional groups may be the same ordifferent than the internal functionalization 108. For example, theexternal functionalization 110 may attract polynucleotides 102 to theexterior surface of the self-assembling vesicle 100. Externalfunctionalization 110 may also create a covalent bond or non-covalentinteraction with an encapsulating layer (shown in FIG. 2) that surroundsthe self-assembling vesicle 100.

External groups may also control the pore size of the membrane 106 andblock large polynucleotides 102 from entering. For example, largeexternal groups may create steric hindrance that preventspolynucleotides above a certain length from entering a membrane pore.Thus, in this way, external functionalization 110 can act as asize-exclusion gate. Size-specific access to pores may also becontrolled by using external groups that can undergo conformationalmodifications. For example, click chemistry modifiers that cross-linkunder specific conditions such as addition of a metal catalyst (e.g.,copper(I)-catalyzed azide/alkyne cycloaddition (CuAAC)).

One or more types of stabilizers may be associated with theself-assembling vesicle 100. One type of stabilizer is a nucleic-acidstabilizer 112 that stabilizes the polynucleotides 102 inside theself-assembling vesicle 100. The nucleic-acid stabilizer 112 may be asalt such as a chloride salt (e.g., sodium chloride, calcium chloride,magnesium chloride, lanthanum chloride, etc.) a phosphate salt (e.g.,calcium phosphate), a nitrate (e.g., calcium nitrate, magnesium nitrate,lanthanum nitrate, etc.), a carbonate (e.g., calcium carbonate,magnesium carbonate, lanthanum carbonate, etc.), sodium benzoate,magnesium sulfate, lanthanum bromide, or a mixture thereof. Cations inthe salt may be selected from the group comprising calcium, magnesium,and lanthanum. Anions in the salt may be selected from the groupcomprising chloride, fluoride, nitride, bromide, iodide, phosphate,carbonate, nitrate, perchlorate, iodate, and bromate. Chelators such asethylenediaminetetraacetic acid (EDTA) and diaminocyclohexanetetraacetate are another type of suitable nucleic-acid stabilizer 112.Additionally, polysaccharides such as dextran, raffinose, and trehalosecan function as nucleic-acid stabilizers 112. Nucleic acids may also bestabilized by sugar alcohols such as mannitol and polyamines such asspermine. Furthermore, commercially available chemical stabilizers suchas GenTegra™-DNA available from GenTegra® LLC and DNAstable® availablefrom Biomatrica® may also be used as the nucleic-acid stabilizer 112.

A membrane stabilizer 114 may be used to stabilize the membrane 106 ofthe self-assembling vesicle 100. The membrane stabilizer 114 may be asmall molecule such as a radical scavenger that protects theself-assembling vesicle 100 from ultraviolet radiation and/or peroxides.Protective molecules that may be used as the membrane stabilizer 114include butylated hydroxytoluene (“BHT” which is a lipophilic organiccompound, a derivative of phenol), diphenylamine (an organic compoundwith the formula (C₆H₅)₂NH), phosphite esters (organophosphoruscompounds with the formula P(OR)₃), benzophenone (a UV-blocking organiccompound with the formula ([phenyl]C₆H₅)₂CO, abbreviated Ph₂CO), and thelike.

The precursors 104 may also contain components that form pores 116 inthe self-assembling vesicle 100. Pores 116 may be formed from a numberof different natural or synthetic molecules such as alpha-haemolysin,DNA origami nanopores, cyclodextrin, gramicidin, and calixarene. Manybiological pores or synthetic channels will spontaneously embed in themembrane 106 during formation of the self-assembling vesicle 100.

The pores 116 may also be formed by electroporation. Electroporationforms temporary pores in the membrane 106 as a result of applying anelectrical field that increases membrane permeability. Electroporationworks by passing thousands of volts across a distance of one to twomillimeters through a solution of suspended vesicles. Subjecting themembrane 106 to a high-voltage electric field results in temporarybreakdown creating pores 116 that are large enough to allowpolynucleotides to enter or leave the self-assembling vesicle 100.

Passage of polynucleotides into and out of the self-assembling vesicle100 may also be regulated by controlling membrane permeability. Even inthe absence of pores 116, the membrane 106 may allow macromolecules(e.g., polynucleotides) to pass through. A combination of precursorsthat react through a “click chemistry” reaction may also be used tochange membrane permeability and selectively adjust the size ofmolecules that can pass through the membrane 106. A click chemistryreaction is one that is wide in scope and easy to perform, uses onlyreadily available reagents, and is insensitive to the presence of oxygenand water.

Molecules that participate in click chemistry reactions may be includedin the membrane 106 and selectively activated to change membranepermeability. In general, greater cross linking among the moleculespresent in the membrane 106 decreases membrane permeability anddecreases the length of polynucleotides that may pass through. Thus, theextent of membrane cross linking can function as a size-specific filterfor polynucleotide uptake. For example, alkynes like cyclooctyne combinewith azides to undergo azide-alkyne Huisgen cycloaddition due to strain.The ratio of the precursors can control the extent of cross linking andsize of molecule that can pass through membrane 106. Copper-catalyzedclick chemistry reactions (e.g., CuAAC) can be controlled by the lengthof time the reactions are allowed to proceed. Longer reaction timegenerally results in the membrane becoming less permeable and loweringthe threshold size of molecules that can enter.

Additional protection may be achieved by forming multiple vesiclemembranes around the polynucleotides 102. After the self-assemblingvesicle 100 is formed, additional precursors for a different type ofvesicle may be added and these precursors may form a second membrane(not shown) around the existing membrane 106. For example, liposomes maybe encapsulated in proteinosomes to create a double membrane of twodifferent vesicle types around the polynucleotides 102. Additionallayers, e.g., a third layer of coacervate membrane, may also be added.Recall that the external functionalization 110 may add functional groupsthat attract polynucleotides 102. Polynucleotides 102 attached orattracted to the exterior of the membrane 106 may be encapsulated by asecond vesicle membrane to create alternating layers of vesiclemembranes and polynucleotides 102. This may be repeated for vesicleswith more than two membrane layers.

FIG. 2 shows the self-assembling vesicle 100 surrounded by anencapsulating layer 200. The encapsulating layer 200 is not anadditional self-assembling membrane as described above, but is astructural, protective layer formed from a non-reactive material such assilica, titanium, gold, a thin film wax (e.g. lipid), cyanate-derivedester resins, biocompatible and biodegradable polysaccharide hydrogelssuch as those made from chitosan-tripolyphosphate, nanocellulosecomposites, fibroin (i.e., silk protein) or other polymeric or monomericmolecule. The types of polymers that may be used in the encapsulatinglayer 200 include polyester-based polymers, diblock co-polymers, andtri-block co-polymers. Specific examples of suitable polymers includepolylactic acid (PLA), polyethylene glycol (PEG), PEG-PDLLA, PEG-PLA,Poly(methyl methacrylate) (PMMA),poly(N-(2-hydroxypropyl)methacrylamide) (HPMA), orpoly(lactic-co-glycolic acid) (PLGA). Metals such as titanium or goldcan provide a thin film around the self-assembling vesicle 100 thatprevents entry of damaging chemicals, light, and moisture. Multipleencapsulating layers (not shown) such as a layer of silica furthersurrounded by layer titanium are also possible.

Silica, also known as, silicon dioxide, silicic acid, or silicic acidanhydride is an oxide of silicon with the chemical formula SiO₂.Encapsulation in silica can hermetically seal the polynucleotides 102under glass thereby protecting them from ROS, chemical attack, hightemperatures, and humidity. An encapsulating layer 200 made of silicamay be formed by contacting the self-assembling vesicle 100 with asilica-containing compound such as one or more tetra alkoxysilanes. Inan implementation, the tetra alkoxysilane(s) may have an alkoxy chainwith between one and five carbon atoms. For example, the tetraalkoxysilane may be tetramethyl orthosilicate, tetraethyl orthosilicate(TEOS), tetrapropyl orthosilicate, tetrabutyl orthosilicate, tetrapentylorthosilicate, or mixtures thereof. TEOS is the most prevalent alkoxideof silicon. It is a chemical compound with the formula Si(OC₂H₅)₄. TEOSis a colorless liquid that degrades in water. TEOS is the ethyl ester oforthosilicic acid, Si(OH)₄. TEOS easily converts to silicon dioxide uponthe addition of water. This hydrolysis reaction is an example of asol-gel process. The side product is ethanol. The reaction proceeds viaa series of condensation reactions that convert the TEOS molecule into amineral-like solid via the formation of Si—O—Si linkages. Rates of thisconversion are sensitive to the presence of acids and bases, both ofwhich serve as catalysts.

The silica-containing compound may be in an aqueous solution and mixedwith dried self-assembling vesicles 100 for an extended period of timesuch as several hours or days. The mixing may be facilitated by use of ashaker. In an implementation, the mixing may be performed for 12 hours,24 hours, 48 hours, 72 hours, 96 hours, or longer. Longer periods ofmixing may result in a thicker silica shell forming around theself-assembling vesicle 100. In some implementations, the silica layerformed over the self-assembling vesicles 100 by this method may beapproximately 10 nm thick.

An encapsulating layer 200 of silica may also be formed by sputterdeposition of silicon dioxide. Sputter deposition is a physical vapordeposition (PVD) method of depositing thin films by ejecting sputteringmaterial from a “target” that is a source of the material, thendepositing it onto a “substrate” which in this application would be theself-assembling vesicles 100. Chemical vapor deposition (CVD) may alsobe used to deposit silica onto the self-assembling vesicles 100. CVD isa deposition method used to apply a thin film of solid material to asurface typically under vacuum. For example, with CVD, polycrystallinesilicon may be deposited from trichlorosilane (SiHCl₃) or silane (SiH₄).

Additionally or alternatively, titanium dioxide, TiO₂, or gold may bedeposited on the self-assembling vesicle 100 by CVD, for example, bylow-pressure CVD (LPCVD) which is CVD at sub-atmospheric pressures.Titanium dioxide can form a thin film over the self-assembling vesicle100 which may have a thickness of about 15 nm.

External functionalization on the surface of the self-assembling vesicle100 may form covalent bonds to the material that makes up theencapsulating layer 200. The covalent bond may strengthen theassociation between that encapsulating layer 200 and the self-assemblingvesicle 100. Additionally, during retrieval of the polynucleotides,removal of the encapsulating layer 200 will also remove fragments of themembrane due to the covalent bond. This may increase the ease ofpurifying the polynucleotides when retrieved from storage.

As a form of additional protection or as an alternative to encapsulatingthe self-assembling vesicles 100, the self-assembling vesicles 100 maybe covered with a thin film. The thin film may be a plastic film made ofsynthetic organic polymers such as polyethylene plastic wrap or plasticparaffin film. The self-assembling vesicles 100 may be dried to preventtrapping moisture under the thin film. Covering the self-assemblingvesicles 100 with a thin film does not surround and encapsulate theentirety of the vesicles with the thin film, but rather covers one sideof the self-assembling vesicles while the other side is protected by asubstrate or support material on which the self-assembling vesicles 100are placed.

Illustrative Processes

For ease of understanding, the processes discussed in this disclosureare delineated as separate operations represented as independent blocks.However, these separately delineated operations should not be construedas necessarily order dependent in their performance. The order in whichthe process is described is not intended to be construed as alimitation, and any number of the described process blocks may becombined in any order to implement the process or an alternate process.Moreover, it is also possible that one or more of the providedoperations is modified or omitted.

FIG. 3 shows process 300 for storing polynucleotides in self-assemblingvesicles. Process 300 may be implemented using the structures shown inFIGS. 1 and 2.

At operation 302, self-assembling vesicle precursors are mixed with asolution of polynucleotides. The mixing may be performed manually or byuse of automated liquid-handling equipment. The self-assembling vesicleprecursors may be amphipathic lipids such as phospholipids, colloidparticles, conjugated protein-polymers such as BSA-PNIPAAm, oppositelycharged macroions, polyesters, polysaccharides, or the like. Thepolynucleotides may be in a solution of annealing buffer (e.g., at aconcentration of 50 μg/ml). The annealing buffer may be any standardbuffering solution for polynucleotides such as 400 mM Tris-HCl, 500 mMNaCl, and 100 mM MgCl₂. The polynucleotides may have a length of about100-300 bp such as a length of approximately 150 bp. In animplementation, the polynucleotides may encode digital information.

At operation 304, one or more stabilizers are added. The stabilizers maybe nucleic-acid stabilizers that stabilize the polynucleotides, membranestabilizers that stabilize the membranes of the self-assemblingvesicles, and an encapsulating layer that contacts the outside of theself-assembling vesicles. The nucleic-acid stabilizer may be any type ofknown stabilizer for DNA or RNA such as salts, chelators,polysaccharides, polyamines, or commercial stabilizer formulations. Themembrane stabilizers may be any type of molecular structure thatstabilizes the membrane of the vesicles. The particular membranestabilizer will vary with the type of vesicle. The encapsulating layermay be formed from a non-reactive material such as silica, titanium, ora polymer. Any number of different types of stabilizers may be combined.For example, the polynucleotides may be stabilized with a nucleic-acidstabilizer and the self-assembling vesicle may be stabilized withmembrane stabilizers and surrounded by an encapsulating layer.

At operation 306, the mixture of polynucleotides, self-assemblingvesicle precursors, and stabilizers are mechanically agitated.Mechanical agitation may be performed by vortexing, sonication, or othermeans. Mechanic agitation may increase the speed of vesicle formation.DNA or RNA shearing due to vortexing or sonication is unlikely when thelength of the polynucleotide strands is relatively short (i.e., <500 bp)as is the case for artificially synthesized polynucleotides. Mild heat(e.g., about 60-80° C.) may also be used to increase the rate of vesicleformation.

At operation 308, the self-assembling vesicles are dried. Addition ofsalts as nucleic-acid stabilizers may enhance desiccation of thevesicles and polynucleotides contained within. The dry vesicles may takethe form of a powder that can be transferred to one or more containers,spotted on a flat surface, or otherwise manipulated in powder form.Spotting onto a flat surface may be used to place portions of the driedvesicles at known, addressable locations that can be later identifiedbased on the position of the spotting on the flat surface.Alternatively, spotting may be performed while the vesicles are stillwet and then they may be dried after.

Any suitable drying technique may be used. In an implementation, dryingmay be performed by use of a centrifugal evaporator or vacuumcentrifuge. A centrifugal evaporator includes a vacuum pump connected toa centrifuge chamber in which samples are placed. The system works bylowering the pressure in the centrifuge system insert—as the pressuredrops so does the boiling point of the solvent(s) in the system. Whenthe pressure is sufficiently low that the boiling points of the solventsare below the temperature of the sample holder, then they boil. Thisenables solvent to be rapidly removed while the samples themselves arenot heated to damaging temperatures. The samples may be processed atroom temperature or heated in the centrifugal evaporator to around25-50° C. Spray drying is another technique that is suitable for dryingthe self-assembling vesicles. Spray drying is the process of convertinga mixture in its liquid form to a powder. This is done by removing themoisture component from the liquid solution. The solution, sometimescalled an emulsion, is sprayed through a nozzle into a chamber thatsimultaneously has hot air being blown into it. Drying may also beperformed by lyophilization (“freeze drying”).

FIG. 4 shows process 400 for obtaining polynucleotides stored invesicles with self-assembling membranes that are protected by anencapsulating layer.

At operation 402, the protective encapsulating layer surrounding theself-assembling vesicle is etched. The technique used for removing theencapsulating layer depends on the materials used in the encapsulatinglayer. Silica or metal encapsulating layers can be removed by etching.The etching may be performed by contacting the self-assembling vesiclewith an etching solution. The specific etching solution will be selectedbased on the type of encapsulating layer. Removing the encapsulatinglayer will expose the self-assembling vesicle.

Silica dissolves rapidly in fluoride-containing solutions (forming SiF₆²⁻). Thus, an etching buffer containing fluoride may be used to dissolvethe silica. The compatibility of dilute etching solutions and DNA (DNAis not affected by F⁻ ions) has previously been shown. The etchingbuffer may be a mixture of a buffering agent such as ammonium fluoride(NH₄F) and hydrofluoric acid (HF). In an implementation, the etchingbuffer may be prepared by dissolving 0.23 g of ammonium hydrogendifluoride (NH₄FHF, puriss, Sigma-Aldrich, cat. no. 30101) in 5 mL ofH₂O and 0.19 g of NH₄F (puriss, Sigma-Aldrich, cat. no. 30101) in 5 mLof H₂O then mixing the two solutions together. Thus, a buffered hydrogenfluoride solution is one suitable etching solution.

A titanium encapsulating layer may also be etched with hydrogenfluoride. For example, an etching solution for titanium may be made from9 V/V % hydrofluoric acid, 12 V/V % nitric acid, and distilled water. Agold encapsulating layer may be etched with potassium cyanide (KCN).

Etching of the protective encapsulating layer may be performed byshaking the self-assembling vesicle in the presence of the etchingsolution. In an implementation, the shaking may be performed forapproximately two minutes at about 900 RPM. No matter how performed,etching may be followed by a washing or cleaning step in which theetched material and etching solution are removed.

At operation 404, the self-assembling vesicle is resuspended insolution. The solution may be in an aqueous solution such as a bufferedsolution. It may be the same solution that the self-assembling vesicleprecursors were stored in prior to vesicle formation.

At operation 406, the membrane of the self-assembling vesicle isdisrupted. The specific technique used for disrupting the membrane willdepend on the type of vesicle. Many biological membranes that can beused to form vesicles may be disrupted with ultrasonication.Ultrasonification uses high-frequency ultrasound (18 kHz−1 MHz) causedby rapid sinusoidal movement of a probe within a liquid. Ultrasonicationproduces cavitation phenomena when acoustic power inputs aresufficiently high to allow the production of multiple microbubbles atnucleation sites in the fluid. The bubbles grow during the rarefyingphase of the sound wave, then are collapsed during the compressionphase. On collapse, a violent shock wave passes through the medium. Thewhole process of gas bubble nucleation, growth, and collapse due to theaction of intense sound waves is called cavitation. The collapse of thebubbles converts sonic energy into mechanical energy in the form ofshock waves equivalent to several thousand atmospheres (300 MPa)pressure. This energy imparts motions to the membrane of theself-assembling vesicle which disintegrates.

Besides ultrasonification, enzymes or other chemicals may be used todisrupt the membrane of the self-assembling vesicle. The membranes ofsome types of self-assembling vesicles may be disrupted by the additionof enzymes such as esterases or lipases that hydrolyzes the membranecomponents. An esterase is a hydrolase enzyme that splits esters into anacid and an alcohol in a chemical reaction with water called hydrolysis.A lipase is any enzyme that catalyzes the hydrolysis of fats (lipids).Lipases are a subclass of the esterases. Non-enzyme chemicals thatdegrade the membrane components may also be used to disrupt someself-assembling vesicles. The specific enzyme or chemical used can beselected based on the composition of the vesicle membrane.

At operation 408, the polynucleotides are purified from the remnants ofthe encapsulating layer, the physical membrane, and other material insolution. Purification removes remnants such as silica, membranefragments, salts, stabilizers, and other compounds from the solutionleaving “clean” polynucleotides that are ready for further processing.If the encapsulating layer is covalently bound to the membrane throughexternal functional groups that were present on the self-assemblyvesicle, then etching the protective layer at 402 may also remove theremnants of the membrane.

If the polynucleotides are bound to internal functional groups on themembrane of the self-assembling vesicle, purification may includedisrupting this association to separate the polynucleotides from themembrane fragments. A release solution may be used to release thepolynucleotides from internal functional groups such as, for example, bydisplacing the polynucleotides. For example, if the internal functionalgroups are polycationic molecules that form electrostatic attachment tothe polynucleotides, the release solution may include poly(acrylic acid)sodium (PAS) which is a polyanionic molecule that displaces thepolynucleotides. This release solution may be made by diluting 40 mg ofPAS in 50 mL of ultrapure water to a concentration of 0.8 mg/mL andmixing that with sodium chloride diluted in ultrapure water untilsaturation at room temperature in a ratio of PAS:NaCl=20:80. As usedherein, “ultrapure water” refers to water having a purity such thatresistivity is 18.2 MΩ·cm at 25° C. or “type 1” water as specified inISO (International Organization for Standards) 3696. One source ofultrapure water is Milli-Q™ water available from Millipore Corporation.

Other techniques for separating the polynucleotides from internalfunctional groups may be used depending on the type of functional group.For example, if the internal functionalization is streptavidin, shortincubation in an aqueous solution above 70° C. will break theinteraction between biotinylated polynucleotides and streptavidinallowing for purification of the polynucleotides.

Many techniques for purifying DNA are known to those of ordinary skillin the art. For example, phenol-chloroform extraction followed byethanol precipitation, ethanol precipitation alone, silica column-basedkit such as the QIAquick PCR purification kit (Qiagen, cat. no. 28104),anion exchange, magnetic beads that bind polynucleotides in apH-dependent manner, or other technique.

At 410, the polynucleotides are sequenced. The polynucleotides may besequenced with any sequencing technology such as sequencing-by-synthesisor Nanopore sequencing. Prior to sequencing, the polynucleotides may beamplified by polymerase chain reaction (PCR) which increases the amountof DNA available. If the polynucleotides encode digital information, thenucleotide base sequence obtained from sequencing the polynucleotidesmay be decoded to recover the digital information.

EXAMPLES

In the following examples, the “sample DNA” was a 150 bp double-strandedDNA molecule with an arbitrary sequence. The sample DNA was obtainedfrom Integrated DNA Technologies (IDT), PCR amplified and purified usingstandard techniques. In these examples, every copy of the sample DNA hadthe same sequence, but in an actual implementation for storage ofdigital information there would be many different sequences representedin a pool of DNA molecules.

FIG. 5 is a fluorescence microscope image of coacervate-DNA conjugates.The white or light-colored spots on the image indicate colocalization ofthe DNA within the coacervates. The coacervates were formed from amixture of poly-T oligonucleotides (ssDNA molecules with approximately20 thymine bases) and poly-L-lysine (poly K) both obtained from IDT. Thesample DNA was fluorescently labeled on its 5′ end with 0.5 μL of a 100μM stock cyanine 5 (Cy5)-labeled oligonucleotide. Cy5 is a fluorescentdye that belongs to the Cyanine family of synthetic polymethine dyes.Cy5 is reactive, water-soluble, and has an absorbance maximum of 649 nmand an emission maximum of 670 nm.

First, 9 μL of 1 mM poly-T was mixed with the sample DNA. Thisoligonucleotide mixture was vortexed briefly. Next, 1 μL of 10 mM ofpoly-L-lysine was added to the oligonucleotide mixture to formcoacervates. This mixture was again vortexed to promote coacervateformation. The coacervates were visualized on an Olympus BX53Mmicroscope equipped with a Cy5™ Filter Cube Set (available from EdmundOptics® stock #67-010-OLY) showing that the sample DNA is concentratedin the spherical coacervates.

FIG. 6 is a scanning electron microscope (SEM) image of coacervatesencapsulated in silicon dioxide. The coacervates were created by mixing89.1 μL of 1 mM poly-T, 9.9 μL of 10 mM poly-L-lysine, and 4.95 μL ofthe sample DNA followed by vortexing. The sample DNA was labeled withfluorescein amidite (FAM). FAM is a synthetic equivalent of fluoresceindye. Thirty μL of this mixture was diluted in 470 μL of DNase and RNasefree molecular grade water. To provide silica encapsulation, 4.5 μL ofreagent grade 98% TEOS solution (Sigma-Aldrich, catalog number 131903)was added to the diluted coacervate mixture. The TEOS was allowed toreact with the coacervates for four days at room temperature withshaking at 900 rpm. The SEM image generated by a Sirion XL30 scanningelectron microscope shows silica-encapsulated coacervates. Thesilica-encapsulated coacervates are both smaller and appear to have moreuniform sizing than the coacervates shown in FIG. 5.

FIG. 7 is an image of an electrophoretic gel showing that encapsulationof coacervates with TEOS protects from degradation by bleach. The DNAcontrol is the 150 bp sample DNA without any processing or protection.DNA full process is DNA prepared without addition of the coacervateprecursors, TEOS, or tris-(methoxy)aminopropylsilane (TMAPS). Thecoacervates sample is coacervates containing the sample DNA without anyadditional protective layer. The coacervates sample was prepared usingthe procedure described for FIG. 6 but without addition of TEOS. TheTEOS/TMAPS sample was also prepared using the procedure of FIG. 6 withthe modification of including 0.5 μL of 50% 3-(trimethylammonium)propylfunctionalized silica (TMAPS) solution in ethanol during the fourdays of shaking. TMAPS is an abbreviation forN-Trimethoxysilylpropyl-N,N,N-trimethylammonium. The chloride salt ofTMAPS has the formula C₉H₂₄ClNO₃Si and is available in 50% methanol fromFisher Scientific company L.L.C. (catalog number AAH6641414). The TEOSsample was prepared according to the procedure described for FIG. 6.

All samples were exposed to a 5% bleach solution for 30 minutes. Bleachis known to degrade DNA and is a source of strong reactive oxygenspecies (ROS). All samples other than the DNA control were etched forapproximately five minutes with 50 μL of a hydrofluoric acid etchingsolution prepared by dissolving 0.23 g of ammonium hydrogen difluoride(NH₄FHF, puriss, Sigma-Aldrich, catalog number 30101) in 5 mL of H₂O and0.19 g of NH₄F (puriss, Sigma-Aldrich, catalog number 30101) in 5 mL ofH₂O then mixing the two solutions together. The DNA from all samplesother than the DNA control were cleaned by mixing with 50 μL of 0.3 MNaOAc to remove the poly-L-lysine that formed the coacervates.

All samples were run on an electrophoretic gel. Only the sample ofcoacervates encapsulated in TEOS had a band at 150 bp. DNA in the othersamples was apparently fully degraded by exposure to bleach. Withoutbeing bound by theory, it is believed that the combination of TEOS andTMAPS forms a different crystal structure than TEOS alone and that theTEOS/TMAPS crystal structure provides less protection.

Illustrative Embodiments

The following clauses described multiple possible embodiments forimplementing the features described in this disclosure. The variousembodiments described herein are not limiting nor is every feature fromany given embodiment required to be present in another embodiment. Anytwo or more of the embodiments may be combined together unless contextclearly indicates otherwise. As used herein in this document “or” meansand/or. For example, “A or B” means A without B, B without A, or A andB. As used herein, “comprising” means including all listed features andpotentially including addition of other features that are not listed.“Consisting essentially of” means including the listed features andthose additional features that do not materially affect the basic andnovel characteristics of the listed features. “Consisting of” means onlythe listed features to the exclusion of any feature not listed.

Clause 1. An article of manufacture for storing polynucleotidescomprising: a self-assembling vesicle containing polynucleotides; and astabilizer.

Clause 2. The article of manufacture of clause 1, wherein theself-assembling vesicle is a protocell, a liposome, a micelle, acolloidosome, a proteinosome, or a coacervate.

Clause 3. The article of manufacture of clause 1 or 2, wherein theself-assembling vesicle further comprises pores that excludepolynucleotides above a threshold size.

Clause 4. The article of manufacture of clause 1-3, wherein an internalsurface of the self-assembling vesicle is functionalized with functionalgroups that attract the polynucleotides.

Clause 5. The article of manufacture of clause 4, wherein the functionalgroups comprise a nucleic acid sequence complementary to a portion of asequence of the polynucleotides.

Clause 6. The article of manufacture of clause 1-5, wherein thepolynucleotides are present in the self-assembling vesicle at a higherconcentration than in a solution containing the polynucleotides prior toformation of the self-assembling vesicle.

Clause 7. The article of manufacture of clause 1-6, wherein thestabilizer is a nucleic-acid stabilizer present inside theself-assembling vesicle.

Clause 8. The article of manufacture of clause 7, wherein thenucleic-acid stabilizer comprises at least one of sodium benzoate, achloride salt, a phosphate salt, ethylenediaminetetraacetic acid (EDTA),diaminocyclohexane tetraacetate, dextran, raffinose, trehalose,mannitol, or spermine.

Clause 9. The article of manufacture of clause 1-8, wherein thestabilizer is an encapsulating layer contacting the outside of theself-assembling vesicle.

Clause 10. The article of manufacture of clause 9, wherein theencapsulating layer comprises silicon dioxide, titanium dioxide, gold, athin-film wax, an ester resin, a polysaccharide hydrogel, PEG, PLA,PEG-PDLLA, PEG-PLA, PMMA, HPMA, PLGA, chitosan-tripolyphosphate,nanocellulose composites, or silk fibroin.

Clause 11. The article of manufacture of clause 9, wherein an exteriorof the self-assembling vesicle is functionalized to covalently bond withthe encapsulating layer.

Clause 12. A method for concentrating and stably storing polynucleotidescomprising: mixing self-assembling vesicle precursors with a solutioncontaining the polynucleotides; and adding a stabilizer, wherein a firstconcentration of the polynucleotides inside a vesicle formed from theself-assembling vesicle precursors is higher than a second concentrationof the polynucleotides in the solution.

Clause 13. The method of clause 12, wherein the self-assembling vesicleprecursors are amphipathic lipids, colloid particles, conjugatedprotein-polymers, oppositely charged macroions, polyesters, orpolysaccharides.

Clause 14. The method of clause 12 or 13, wherein the stabilizer is anucleic-acid stabilizer that associates with and stabilizes thepolynucleotides, a membrane stabilizer that associates with andstabilizes an external surface of the vesicle, or a protectivestructural layer that encapsulates the vesicle.

Clause 15. The method of clause 12-14, further comprising mechanicallyagitating a mixture of the self-assembling vesicle precursors and thesolution containing polynucleotides.

Clause 16. The method of clause 12-15, further comprising drying thevesicle containing the polynucleotides.

Clause 17. A method of accessing polynucleotides stored in aself-assembling vesicle comprising: etching a protective encapsulatinglayer surrounding the self-assembling vesicle to expose theself-assembling vesicle; resuspending the self-assembling vesicle insolution; disrupting a membrane of the self-assembling vesicle; andpurifying the polynucleotides.

Clause 18. The method of clause 17, wherein etching the protectiveencapsulating layer comprises contacting the self-assembling vesiclewith a hydrogen fluoride solution.

Clause 19. The method of clause 17 or 18, wherein disrupting themembrane comprises ultrasonication, contacting the membrane with ahydrolase, or contacting with membrane with a chemical that degrades oneor more membrane components.

Clause 20. The method of clause 17-19, further comprising sequencing thepolynucleotides and decoding a sequence of nucleotide bases obtainedfrom the sequencing to recover digital information stored in thepolynucleotides.

CONCLUSION

Although the subject matter has been described in language specific tostructural features and/or methodological acts, it is to be understoodthat the subject matter defined in the appended claims is notnecessarily limited to the specific features or acts described above.Rather, the specific features and acts are disclosed as example forms ofimplementing the claims.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.The terms “based on,” “based upon,” and similar referents are to beconstrued as meaning “based at least in part” which includes being“based in part” and “based in whole,” unless otherwise indicated orclearly contradicted by context. The terms “portion,” “part,” or similarreferents are to be construed as meaning at least a portion or part ofthe whole including up to the entire noun referenced. As used herein,“approximately” or “about” or similar referents denote a range of ±10%of the stated value.

Certain embodiments are described herein, including the best mode knownto the inventors for carrying out the invention. Of course, variationson these described embodiments will become apparent to those of ordinaryskill in the art upon reading the foregoing description. Skilledartisans will know how to employ such variations as appropriate, and theembodiments disclosed herein may be practiced otherwise thanspecifically described. Accordingly, all modifications and equivalentsof the subject matter recited in the claims appended hereto are includedwithin the scope of this disclosure. Moreover, any combination of theabove-described elements in all possible variations thereof isencompassed by the invention unless otherwise indicated herein orotherwise clearly contradicted by context.

Furthermore, references have been made to publications, patents and/orpatent applications (collectively “references”) throughout thisspecification. Each of the cited references is individually incorporatedherein by reference for its particular cited teachings as well as forall that they disclose.

1. An article of manufacture for storing polynucleotides comprising: aself-assembling vesicle formed from self-assembling vesicle precursorsand containing polynucleotides; a stabilizer; and a protectiveencapsulating layer surrounding the self-assembling vesicle, wherein theprotective encapsulating layer comprises silicon dioxide or titaniumdioxide.
 2. The article of manufacture of claim 1, wherein theself-assembling vesicle is a protocell, a liposome, a micelle, acolloidosome, a proteinosome, or a coacervate.
 3. The article ofmanufacture of claim 1, wherein the self-assembling vesicle precursorscomprise amphipathic lipids, colloid particles, conjugatedprotein-polymers, oppositely charged macroions, polyesters, orpolysaccharides.
 4. The article of manufacture of claim 1, furthercomprising two types of self-assembling vesicle precursors and theself-assembling vesicle comprises a double membrane.
 5. The article ofmanufacture of claim 1, wherein the self-assembling vesicle furthercomprises pores that exclude polynucleotides above a threshold size. 6.The article of manufacture of claim 5, wherein the pores comprisealpha-haemolysin, DNA origami nanopores, cyclodextrin, gramicidin, orcalixarene.
 7. The article of manufacture of claim 1, wherein aninternal surface of the self-assembling vesicle is functionalized withfunctional groups that attract the polynucleotides.
 8. The article ofmanufacture of claim 7, wherein the functional groups comprise a nucleicacid sequence complementary to a portion of a sequence of thepolynucleotides.
 9. The article of manufacture of claim 7, wherein thefunctional groups comprise polycationic molecules that formelectrostatic attachment to the polynucleotides.
 10. The article ofmanufacture of claim 7, wherein the functional groups comprisestreptavidin and the polynucleotides comprise biotinylatedpolynucleotides.
 11. The article of manufacture of claim 1, wherein thepolynucleotides are present in the self-assembling vesicle at a higherconcentration than in a solution containing the polynucleotides prior toformation of the self-assembling vesicle.
 12. The article of manufactureof claim 1, wherein the stabilizer is a nucleic-acid stabilizer presentinside the self-assembling vesicle.
 13. The article of manufacture ofclaim 12, wherein the nucleic-acid stabilizer comprises at least one ofsodium benzoate, a chloride salt, a phosphate salt,ethylenediaminetetraacetic acid (EDTA), diaminocyclohexane tetraacetate,dextran, raffinose, trehalose, mannitol, or spermine.
 14. The article ofmanufacture of claim 1, wherein the stabilizer is a membrane stabilizerthat associates with and stabilizes an external surface of theself-assembling vesicle.
 15. The article of manufacture of claim 14,wherein the membrane stabilizer is butylated hydroxytoluene (BHT),diphenylamine, a phosphite ester, or benzophenone.
 16. The article ofmanufacture of claim 1, wherein the protective encapsulating layercomprises silicon dioxide.
 17. The article of manufacture of claim 1,wherein the protective encapsulating layer comprises titanium dioxide.18. The article of manufacture of claim 1, wherein an exterior of theself-assembling vesicle is functionalized to covalently bond with theprotective encapsulating layer.
 19. An article of manufacture forstoring polynucleotides comprising: a self-assembling coacervate formedfrom poly-L-lysine and containing polynucleotides; poly-Toligonucleotides; and a protective encapsulating layer surrounding theself-assembling coacervate, wherein the protective encapsulating layercomprises silicon dioxide.
 20. The article of manufacture of claim 19,wherein the polynucleotides comprise synthetic DNA having a sequencethat encodes digital information.